Degradable polyesters, a mixed culture of microorganisms for degrading these polyesters, and methods for making these substances

ABSTRACT

Degradable polyesters useful in packaging, packing, agricultural, biomedical, and other applications are made by reacting amine-protected glutamic acid with diols or epoxy compounds. The polyesters include a thermoplastic main chain aliphatic polyester, a thermoset heterochain polyester and a thermoset heterochain aromatic polyester. Each of these polyesters can be hydrolyzed into monomers using a biological catalyst such as the enzyme lipase. The thermoplastic main chain aliphatic polyester and the thermoset heterochain polyester can be degraded to respiratory gases and biomass with a mixed culture of  Rhizopus chinesis, Rhizopus delemar, Penecillium pinophilum, Aspergillus niger  and  Pseudomonas aeruginosa  microorganisms. This mixed culture of microorganisms can also be used to degrade other polyesters containing hydrolyzable backbone polyesters.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 09/358,348 filed Jul. 21,1999, which claims benefit of Ser. No. 60/067,514 filed Dec. 4, 1997,and a division of Ser. No. 09//204,338 filed Dec. 2, 1998 now U.S. Pat.No. 5,990,266.

BACKGROUND OF THE INVENTION

The present invention relates generally to polymeric materials and moreparticularly to polyesters, as well as methods for making and degradingpolyesters. The invention also relates to a mixed culture ofmicroorganisms that is able to degrade polymers containing ahydrolyzable backbone polyester and a method for making this mixedculture of microorganisms.

The majority of plastic materials available today are made frompetroleum. Such petroleum-based plastics are difficult to degrade,biologically or otherwise. Because of the extensive use ofpetroleum-based plastics by both consumers and industry, a considerableamount of waste is created. This causes ecological problems because thewaste is stored in landfills and other waste disposal systems andaccumulates without degradation. Furthermore, petroleum is an expensivecomponent of plastic.

Biodegradable polymers have been synthesized to provide alternatives topetroleum-based plastics. They are often synthesized from starch, sugar,natural fibers or other organic biodegradable components in varyingcompositions. However, such biodegradable polymers often lack desirablephysical characteristics, and this limits their use to specificsituations. Many biodegradable polymers are blended polymers orcomposite polymers and thus do not have uniform mechanical properties.Still further, most known biodegradable polymers are aliphaticpolyesters that have low softening points (T_(m)), which prevents theiruse as a material in a variety of fields.

In order to overcome the deficiencies found with conventional plasticsand biodegradable polymers, a degradable polymer having enhancedstrength and flexibility and a method for making such a degradablepolymer are needed for a variety of applications. Still further, apolymer that is made from organic substances rather than petroleum-basedcompounds and a method for making this polymer are needed. In addition,certain formulations of this polymer should be biodegradable. A mixedculture of microorganisms that can be used to degrade the polymers ofthe present invention and other polymers and a method for making thismixed culture are also needed.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide degradable polymershaving uniform mechanical properties and high softening points allowingthem to replace petroleum-based plastics for various applications and amethod for making such polymers in order to avoid the use ofpetroleum-based plastics and the waste generated by using suchcompounds.

It is a further object of the present invention to provide a mixedculture of microorganisms that can completely degrade differentpolyesters into respiratory gases and biomass and a method for makingthis mixed culture so that waste can be removed from the environment.

Another object of the present invention is to provide non-toxicdegradable polymers and a method for making these polymers so that theycan be used in the human body as drug-loaded matrices, surgical threadand surgical implants.

Still another object of the present invention is to providebiodegradable polymers having physical strength so that they can be usedas a packaging material that can be disposed of by degradation.

According to the present invention, the foregoing and other objects areachieved by a thermoplastic main chain aliphatic polyester, a thermosetheterochain polyester, or a thermoset heterochain aromatic polyester.Each of these polymers are embodiments of the present invention that aremade by reacting various organic compounds with blocked glutamic acid.The thermoplastic main chain aliphatic polyester is made by reactingdiol with blocked glutamic acid. The thermoset heterochain and thermosetheterochain aromatic polyesters are formed by reacting an epoxy compoundwith blocked glutamic acid. All of these polyesters can be hydrolyzedinto monomers when treated with certain biological catalysts such as theenzyme lipase. Furthermore, the thermoplastic main chain aliphaticpolyester and the thermoset heterochain polyester can be completelydegraded into respiratory gases and biomass using certain mixed culturesof microorganisms. One such mixed culture of microorganisms, which is anembodiment of the present invention, is comprised of the mixture ofRhizopus chinesis, Rhizopus delemar, Penecillium pinophilum, Aspergillusniger and Pseudomonas aeruginosa. The polyesters of the presentinvention may be used in medical applications, agriculturalapplications, packaging applications, packing applications, and anyother applications where plastics are used.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned from practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings form a part of this specification and are tobe read in conjunction therewith.

FIG. 1 is a Gel Permeation Chromatography (GPC) graph of thethermoplastic main chain aliphatic polyester as it forms during thepolymerization reaction.

FIG. 2 is a GPC graph of the thermoset heterochain polyester as it formsduring the polymerization reaction.

FIG. 3 is a GPC graph of the thermoset heterochain aromatic polyester asit forms during the polymerization reaction.

FIG. 4 is a GPC graph of the thermoplastic main chain aliphaticpolyester as it undergoes hydrolysis by lipase enzyme.

FIG. 5 is a GPC graph of the thermoset heterochain polyester as itundergoes hydrolysis by lipase enzyme.

FIG. 6 is a GPC graph of the thermoset heterochain aromatic polyester asit undergoes hydrolysis by lipase enzyme.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The polymers of the present invention are a thermoplastic main chainaliphatic polyester, a thermoset heterochain polyester, and a thermosetheterochain aromatic polyester. These polymers have structures whichallow them to degrade. The polymer is hydrolyzed by the secretion ofextracellular enzymes by microorganisms into low molecular weightcompounds. These low molecular weight compounds can be introduced intothe microbial cell for further degradation. All of these polyesters areformed by reacting amine-protected glutamic acid with various monomers.Still further, glutamic acid can be replaced by α-amino dicarboxyllicacids, such as aspartic acid, to produce polymers that are consideredalternate embodiments of the present invention. The polymers that can beformed include polymers of various molecular weights, molecularstructures and physical characteristics.

Glutamic acid is a biodegradable material, according to the in vivopathway of metabolism. For example, L-glutamic acid is an intermediatein the Krebs cycle. It is an inexpensive and abundant amino acid andexists in partucularly high percentages in oilseed proteins, such assoybeans. In some genera of soybeans, about 60% of the seed is protein.Glutamic acid is a major component, about representing 18-22% of theamino acid content of major oilseed proteins. Glutamic acid also can bechemically synthesized or biologically produced (fermentation).Furthermore, there are many inexpensive commercially acceptable methodsavailable for hydrolyzing and separating glutamic acid from the rest ofthe amino acids in the oilseed protein.

Using glutamic acid in polymerization reactions in its natural,unblocked form limits polymerization. When glutamic acid is heated witha monomer for polymerization, the glutamic acid tends to form cycliccompounds (mostly lactam) rather than linear molecular formations, andthis inhibits the progress of polymerization. Therefore, in preparingthe polymers of the present invention, it is first necessary to blockthe functional groups on the glutamic acid molecules, namely the aminegroups, to prevent the formation of cyclic compounds such as lactam.

A number of different blocking agents can be used to block the aminegroup on the glutamic acid. These blocking agents include acyl-typeprotecting groups, urethan protecting groups, alkyl-type protectinggroups, and arylidene-type protecting groups. Alternatively, rather thanusing blocking agents, the amine group can be protonated to block itsfunctionality. Preferably, blocking of the glutamic acid is achieved byreacting it with carbobenzoxy chloride in an aqueous alkaline solutionto form N-carbobenzoxy glutamic acid, which is commonly referred to asZ-glutamic acid. This reaction is inexpensive and provides about a 90%yield of Z-glutamic acid. Alternatively, Z-glutamic acid can be obtainedfrom Aldrich Chemical Company, 1001 West Saint Paul Avenue, Milwaukee,Wis. 53233. Z-glutamic acid has the following structure:

Once the blocked or amine-protected glutamic acid is formed or obtained,the polymers of the present invention can be created by reacting blockedglutamic acid with diol or epoxy compounds. More specifically, a diol,an aliphatic epoxy compound, or an aromatic epoxy compound are reactedwith blocked glutamic acid in the presence of a catalyst to form athermoplastic main chain aliphatic polyester, a thermoset heterochainpolyester, or a thermoset heterochain aromatic polyester, respectively,the polyesters of the present invention. Each of the polyesters of thepresent invention is formed in a one-step reaction.

The polyester of the present invention is generally represented by thefollowing structure, wherein X is a amine protecting group, Y is a diolor an epoxy compound that has been reacted with blocked glutamic acid,and n is an integer:

The thermoplastic main chain aliphatic polyester of the presentinvention may have the following structure when blocked glutamic acid isreacted with a diol, wherein R is an alkyl group, X is a protectinggroup, and n is an integer:

The thermoplastic main chain aliphatic polyester has the followingstructure when ethylene glycol is reacted with Z-glutamic acid, whereinn is an integer:

The thermoset heterochain polyester of the present invention and thethermoset heterochain aromatic polyester of the present invention mayhave one of the following structures (1), (2), or (3), when blockedglutamic acid is reacted with a diepoxy monomer, a triepoxy monomer, anda tetraepoxy monomer, respectively, wherein X is an amine protectinggroup, B is a branch node, is a polymer chain, and n is an integer:

Indications of branch nodes and polymer chains in the above structuresshow that the polyester may have various structures depending upon thebranching within the structure.

The thermoset heterochain polyester has the following structure whendiglycidyl ether of 1,4-Butanediol (BDE) is reacted with Z-glutamicacid, wherein n is an integer:

The thermoset heterochain aromatic polyester has the following structurewhen diglycidyl ether of bisphenol A (DGEBA) is reacted with Z-glutamicacid, wherein n is an integer:

Examples of diols that may be used to form the thermoplastic main chainaliphatic polyester of the present invention include, but are notlimited to, ethylene glycol, propylene glycol, 1,3-butanediol,2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,4-pentanediol,2,4-pentanediol, 1,5-pentanediol, 1,2-hexanediol, 1,5-hexanediol,2,5-hexanediol, 1,6-hexanediol, and 1,7-heptanediol. Preferably, thediol has between about 2 and 14 carbon atoms. Most preferably, it hasbetween about 2 and 8 carbon atoms. When diols having fewer carbon atomsare used in the polymerization reaction, more elastic polymers areformed.

Examples of aliphatic diepoxy components that may be used include, butare not limited to, 1,2,7,8-diepoxyoctane, neopentylglycol diglycidalether, diglycidal ether of 1,4-butanediol, polyglycol diglycidal ether,and cycloaliphatic epoxides. The blocked glutamic acid can also reactwith triepoxy or tetraepoxy structures, which will lead to the formationof a polymer structure having a controlled amount of crosslinking.

Examples of diepoxy compounds that may be used to form the thermosetheterochain aromatic polyester of the present invention include, but arenot limited to, a diglycidyl ether of bisphenol A, diglycidyl ether of4,4′-biphenol, diglycidyl ether of 4,4′-hydroquinone, diglycidyl etherof 1,5-naphthalene diol, diglycidyl ether of 1,6-naphthalene diol, anddiglycidyl ether of 2,7-naphthalene diol. The blocked glutamic acid alsocan be reacted with triepoxy structures, such astriglycidyl-p-aminophenol, or tetraepoxy structures, such asN,N,N′,N′-tetraglycidyl-4,4′-diaminodiphenyl methane and tetraglycidylether of tetraphenolethane. The reaction of blocked glutamic acid withtriepoxy and tetraepoxy structures will lead to a polymer structurehaving a controlled amount of crosslinking.

The polymerization reaction used to form the thermoplastic main chainaliphatic polyester, the thermoset heterochain polyester, and thethermoset heterochain aromatic polyester can proceed without the use ofa catalyst so long as the reactants are heated and allowed to react fora sufficient amount of time. However, preferably, a catalyst is used inthis polymerization reaction. There are a variety of catalysts that maybe used in this reaction, which include, but are not limited to,p-toluene sulfonic acid, sulfuric acid, titanium dioxide xhydrate,titanium tetrachloride, titanium tetrabutoxy, zinc chloride, andstannous oxide.

The blocked glutamic acid is combined with diol in a molar ratio betweenabout 0.5:1 and 1:1.5; preferably, the molar ratio is about 1:1. Theblocked glutamic acid is combined with the diepoxy compound in a molarratio between about 0.5:1 and 2.5:1; preferably, the molar ratio isabout 2:1. The blocked glutamic acid is combined with the triepoxycompound in a molar ratio between about 1:1 and 3.5:1; preferably, themolar ratio is about 3:1. The blocked glutamic acid is combined with thetetraepoxy compound in a molar ratio between about 1:1 and 4.5:1;preferably, the molar ratio is about 4:1. When excess diol or epoxycompound is present, dimers and trimers tend to form. It p-toluenesulfonic acid catalyst is used in this polymerization reaction, about0.025 moles or less of the catalyst should be used per mole of blockedglutamic acid. Other molar ratios of catalyst to blocked glutamic acidmay be preferred if a different catalyst is used.

After the diol or epoxy compound and the blocked glutamic acid aremixed, they are heated and a slow stream of inert gas, such as nitrogenor argon, is bubbled through the molten mixture. Preferably, thisreaction occurs in vacuum conditions. This reaction should take place atabout 90° C. or above for it to occur in a reasonable amount of time.Preferably, the component mixture is heated to between about 90 and 250°C. for about 16 to 24 hours if the diol is a reactant. Most preferably,the component mixture is heated to between about 140 and 170° C. if thediol is a reactant. Preferably, the polymerization reaction occurs atbetween about 90 and 170° C. if the epoxy compound is a reactant. Mostpreferably, the reaction occurs at between about 110 and 130° C. if theepoxy compound is a reactant. After heating the component mixture for atleast about 2 hours at about 90° C. or above, a thermoplastic main chainaliphatic polyester will have formed and may be removed from theapparatus in which the reaction takes place. The resulting polymershould be cooled in a dissicator so as to avoid moisture contact, whichmay cause premature degradation. In addition to having a decreasedreaction time at higher temperatures, the polymer resulting from areaction at higher temperatures will have a higher molecular weight.

The thermoplastic main chain aliphatic polyester of the presentinvention has a number average molecular weight (Mn) between about10,000 and 100,000. This polyester is considered a high quality resin,having good physical properties such as flexibility, impact resistance,the ability to entangle drugs within its matrix, and other desirableproperties.

The thermoset heterochain polyester formed will have a higher molecularweight when the reactants are mixed for a longer time period or when thereaction occurs at a higher temperature. The thermoset heterochainpolyester of the present invention is a somewhat elastic material.Crosslinks and bends in the structure of this polyester improve both itsphysical strength and its biodegradability. The extent of its elasticityis controlled by reactant ratios and reaction conditions. When anapproximately equal molar ratio of blocked glutamic acid to epoxycompound is used, a more flexible and elastic polyester is created.

This thermoset heterochain aromatic polyester has a rigid crystallinestructure. The bulky aromatic groups reduce carbon-carbon rotation,which stiffens the polyester. Thus, it has a stiff and brittle structureat room temperature.

Both the thermoset heterochain polyester and the thermoset heterochainaromatic polyester of the present invention can be considered highquality polymers depending upon how they are being used. Both thesepolymers have gel fractions ranging from about 99.9% to 75% and solfractions ranging from about 0.1% to 25%. Usually, the gel fraction ofthese polymers ranges from about 50 to 60%. Materials having a gelfraction of close to 100% are hard, brittle and strong. Materials havinglower gel fractions are softer, more ductile, more flexible and tend toswell.

The following are examples of the polyesters of the present inventionand methods of making these polyesters. These examples are not meant inany way to limit the scope of this invention:

EXAMPLE 1 Method of Making a Thermoplastic Main Chain AliphaticPolyester

Blocked glutamic acid and ethelene glycol were melt mixed together in abatch reactor under vacuum at 400 mmHg. Ethylene glycol has a lowboiling point and was lost during the early stages of polymerization. Atlater stages of polymerization, ethylene glycol molecules became lessvolatile since they had reacted. P-toluene sulfonic acid catalyst wasadded to the mixture. The blocked glutamic acid and the ethylene glycolwere added in quantities such that the molar ratio of blocked glutamicacid to ethylene glycol to p-toluene sulfonic acid was 1:1.08:0.01. Thereaction was carried out at 110° C. and maintained at this temperaturefor the period of polymerization, which extended for 24 hours. Theexcess diol was stripped from the molten resin by vacuum. The reactionmixture was poured into a reaction bulb and heated in a liquid bath at afixed temperature. A thermoplastic main chain aliphatic polyester of thepresent invention was obtained.

EXAMPLE 2 Method of Making a Thermoplastic Main Chain AliphaticPolyester

Blocked glutamic acid and ethylene glycol were melt mixed together in abatch reactor under vacuum at 400 mmHg. Ethylene glycol has a lowboiling point and was lost during the early stages of polymerization. Atlater stages of polymerization, ethylene glycol molecules became lessvolatile since they had reacted. P-toluene sulfonic acid catalyst wasadded to the mixture. The blocked glutamic acid and the ethylene glycolwere added in quantities such that the molar ratio of blocked glutamicacid to ethylene glycol to p-toluene sulfonic acid was 1:1.08:0.01. Thereaction was carried out at 170° C. and maintained at this temperaturefor the period of polymerization, which extended for 16 hours. Theexcess diol was stripped from the molten resin by vacuum. The reactionmixture was poured into a reaction bulb and heated in a liquid bath at afixed temperature. A thermoplastic main chain aliphatic polyester of thepresent invention was obtained.

EXAMPLE 3 Method of Making a Thermoplastic Main Chain AliphaticPolyester

Blocked glutamic acid and propylene glycol were melt mixed together in abatch reactor under vacuum at 400 mmHg. P-toluene sulfonic acid wasadded as a catalyst. The molar ratio between the blocked glutamic acidto the propylene glycol to the p-toluene sulfonic acid was 1:1.08:0.01.The reaction was carried out at 110° C. and maintained at thistemperature for the period of polymerization, which extended for 22hours. The excess diol was stripped from the molten resin by vacuum. Thereaction mixture was poured into the reaction bulb and heated in aliquid bath at a fixed temperature. A thermoplastic main chain aliphaticpolyester of the present invention was obtained.

EXAMPLE 4 Method of Making a Thermoplastic Main Chain AliphaticPolyester

Blocked glutamic acid and 2,5-hexanediol are melt mixed together in abatch reactor under vacuum at 400 mmHg. P-toluene sulfonic acid is addedas a catalyst. The molar ratio between the blocked glutamic acid to the2,5-hexanediol to the p-toluene to the p-toluene sulfonic acid is1:1.08:0.015. The reaction is carried out at 170° C. and maintained atthis temperature for the period of polymerization, which extends for 16hours. The excess diol is stripped from the molten resin by vacuum. Thereaction mixture is poured into the reaction bulb and heated in a liquidbath at a fixed temperature. A thermoplastic main chain aliphaticpolyester of the present invention is obtained.

EXAMPLE 5 Method of Making a Thermoset Heterochain Polyester

A thermoset heterochain polyester was prepared by melt mixing equivalentamounts of blocked glutamic acid and diglycidal ether of 1,4butanediol(BDE) in the presence of p-toluene sulfonic acid. These components werecombined in a molar ratio of 1:1:0.02, respectively. The polymer mix wasplaced in a pre-heated oven at 110° C. After 15 minutes of heating, thetemperature was raised gradually to the final temperature of 120° C.,and the reaction was maintained at that temperature. The temperature wascontrolled to be within 0.5° C. difference of the set temperature forthe period of polymerization, which extended for 8 hours.

EXAMPLE 6 Method of Making a Thermoset Heterochain Polyester

A thermoset heterochain polyester was prepared by melt mixing equivalentamounts of blocked glutamic acid and diglycidal ether of 1,4butanediol(BDE) in the presence of p-toluene sulfonic acid. These components werecombined in a molar ratio of 1:1:0.02, respectively. The polymer mix wasplaced in a pre-heated oven at 110° C. After 15 minutes of heating, thetemperature was raised gradually to the final temperature of 130° C.,and the reaction was maintained at that temperature. The temperature wascontrolled to be within 0.5° C. difference of the set temperature forthe period of polymerization, which extended for 8 hours.

EXAMPLE 7 Method of Making a Thermoset Heterochain Polyester

A thermoset heterochain polyester is prepared by melt mixing blockedglutamic acid and diglycidal ether of 1,4butanediol (BDE) in thepresence of p-toluene sulfonic acid. These components are combined in amolar ratio of 2:1:0.02, respectively. The polymer mix is placed in apre-heated oven at 110° C. After 15 minutes of heating, the temperatureis raised gradually to the final temperature of 130°0 C., and thereaction is maintained at that temperature. The temperature iscontrolled to be within 0.5° C. difference of the set temperature forthe period of polymerization, which extends for 8 hours.

EXAMPLE 8 Method of Making a Thermoset Heterochain Aromatic Polyester

A thermoset heterochain aromatic polyester was prepared by melt mixingequivalent amounts of blocked glutamic acid and diglycidal ether ofbisphenol A (DGEBA) in the presence of p-toluene sulfonic acid. Thesecomponents were combined in a molar ratio of 1:1:0.02, respectively. Thepolymer mix was placed in a pre-heated oven at 110° C. After 15 minutesof heating, the temperature was raised gradually to the finaltemperature of 120° C., and the reaction was maintained at thattemperature. The temperature was controlled to be within 0.5° C.difference of the set temperature for the period of polymerization,which extended for 5 hours.

EXAMPLE 9 Method of Making a Thermoset Heterochain Aromatic Polyester

A thermoset heterochain aromatic polyester was prepared by melt mixingequivalent amounts of blocked glutamic acid and diglycidal ether ofbisphenol A (DGEBA) in the presence of p-toluene sulfonic acid. Thiscomponents were combined in a molar ratio of 1:1:0.02, respectively. Thepolymer mix was placed in a pre-heated oven at 110° C. After 15 minutesof heating, the temperature was raised gradually to the finaltemperature of 130° C., and the reaction was maintained at thattemperature. The temperature was controlled to be within 0.5° C.difference of the set temperature for the period of polymerization,which extended for 5 hours.

EXAMPLE 10 Method of Making a Thermoset Heterochain Aromatic Polyester

A thermoset heterochain aromatic polyester is prepared by melt mixingblocked glutamic acid and diglycidal ether of bisphenol A (DGEBA) in thepresence of p-toluene sulfonic acid. These components are combined in amolar ratio of 2:1:0.02, respectively. The polymer mix is placed in apre-heated oven at 110° C. After 15 minutes of heating, the temperatureis raised gradually to the final temperature of 130° C., and thereaction is maintained at that temperature. The temperature iscontrolled to be within 0.5° C. difference of the set temperature forthe period of polymerization, which extends for 5 hours.

EXAMPLE 11 Method of Making a Thermoset Heterochain Aromatic Polyester

A thermoset heterochain aromatic polyester is prepared by melt mixingblocked glutamic acid and tetraglycidyl ether of tetraphenolethane withp-toluene sulfonic acid. These components are combined in a molar ratioof 4:1:0.01, respectively. The polymer mix is placed in a pre-heatedoven at 110° C. After 15 minutes of heating, the temperature is raisedgradually to the final temperature of 130° C., and the reaction ismaintained at that temperature. The temperature is controlled to bewithin 0.5° C. difference of the set temperature for the period ofpolymerization, which extends for 10 hours.

Gel Permeation Chromatography (GPC) is used to quantitate changes inmolecular weight distribution of polymers during polymerization anddegradation processes. The increase in molecular weight of the threenovel polymers of the present invention during the polymerizationreactions is shown in FIGS. 1-3.

Each of the polymer types of the present invention, namely, thethermoplastic main chain aliphatic polyester, the thermoset heterochainpolyester, and the thermoset heterochain aromatic polyester, can behydrolyzed into their monomeric units by many biological catalysts. Anesterase enzyme, such as lipase, which is extracted from themicroorganism Rhizopus delemar or other microorganisms, is one exampleof a biological catalyst that may be used to hydrolyze the polyesters.Another biological catalyst that can be used is hog liver esterase. Thebiological catalyst randomly splits the ester bonds of the polyester,hydrolyzing it into low molecular weight compounds. Hydrolysis intomonomers is the first step of degradation. The decline in molecularweight of the three polymers of the present invention as they undergohydrolysis is shown in FIGS. 4-6.

Another step of degradation involves treating these polymers with amixed culture of microorganisms, which is designed to metabolize orbreak down the polymers' structures. In catabolic processes occurring innature, such as the degradation of cellulose and lignin, biodegradationproceeds best using mixed populations of microorganisms and isrestricted or incomplete when a pure culture or a single strain ofmicroorganisms is used. In the same way, mixed cultures were chosen tometabolize the polyesters of the present invention rather than a pureculture. Cellulose or lignin degradation may resemble the degradation ofplastics in a general way but not with respect to enzymological details.Since differences among microorganisms always implies differences inenzymatic activities, in forming the mixed culture of microorganisms ofthe present invention, the mixed culture used to degrade cellulose orlignin was modified by incorporating fungal strains whose enzymaticcapability related to the structure of the polyesters of the presentinvention. The microorganisms (mold and bacteria) chosen are known toproduce specific enzymes. Furthermore, they are known to have adegradable effect on specific classes of polymers and are capable ofmetabolizing aromatic structures.

The mechanism of biodegradation is a composite effect of chemicalreactions that proceed simultaneously on the polymer, accounting for thefact that a second species may be able to flourish after the first hasstarted to wane. Most of the microbial reactions are sequential so thatthe end product of metabolism of a given substrate by one organismbecomes the substrate for another organism. The mixed culture ofmicroorganisms of the present invention is believed to metabolize thesynthesized polymers of the present invention in the same approach.

The mixed culture of microorganisms of the present invention iscomprised of Rhizopus chinesis, Rhizopus delemar, Penecilliumpinophilum, Aspergillus niger, and Pseudomonas aeruginosa. Preferably,these microorganisms are combined in equal proportions. Other fungalstrains have the capability of metabolizing the same polymer structure,and thus, one or more microorganisms may be substituted in the mixedculture. Different species from the same genus may be used in formingthe mixed culture of microorganisms. For instance, Penecilliumbrevicompactum, Penicillium funciculosun, or Penecillium cyclopium maybe added to the mixed culture or may be used a sa substitute forPenecillium pinophilum. Still further, for example, Aspergillusamstelodami, Aspergillus flavus, Aspergillus amstelodami, or Aspergillusversicolor may be added to the mixed culture or may be used as asubstitute for Aspergillus niger. Other Rhizopus and Pseudomonas speciesmay be used in the mixed culture and may be especially effective indegrading polyesters with different chemical structures. The addition ofone or more microorganisms should not have any effect on thedegradability of the various polymers since the chosen mixed culture hasa wide degradable effect over a variety of polyesters with differentchemical structures.

The mold Rhizopus is capable of metabolizing different nutrient carbonsources, such as sugars, ethanol or acetic acid, when each is the onlysource of carbon available. The Rhizopus delemar lipase enzyme randomlysplits ester bonds. It is extracted from Rhizopus species ofmicroorganisms. Different Rhizopus species become a part of the mixedculture of the present invention. Aspergillus niger is effective inmetabolizing different nutrient carbon sources. Aspergillus niger,Pseudomonas aeruginosa, and Penicillium pinophilum are capable ofmetabolizing different polyesters. Pseudomonas putida, pseudomonascruciviae and nocardia restrictus are effective in degrading aromaticstructures. These microorganisms are representative of themicroorganisms used in the mixed culture of the present invention.

The following is an example of a method for preparing a mixed culture ofmicroorganisms. This example is not meant in any way to limit the scopeof this invention.

EXAMPLE 12 A Method for Preparing a Mixed Culture of Microorganisms

The mixture of spores was prepared from separate cultures ofmicroorganisms. Sabouraud dextrose dehydrated media (broth media) wasused as a nutrient for the initial microbial growth. The cultivatedbroth media was prepared by dissolving 30 grams of the dehydrated brothmedia in one liter of distilled water. A volume of 50 ml of thedissolved media was poured into several 150 ml Erlenmeyer flasks. Theprepared flasks were sterilized at 120° C. for 20 minutes. Each fungiused in the mixed culture was cultivated initially in one flask.

The lyophilized preparation of each test organism was inoculated intothe prepared broth liquid media. The prepared cultures were incubated at30° C. in an incubator orbital shaker at 150 rounds per minute (rpm).After fifteen days of growth, 10 ml of the media containing the fungusgrowth was poured into 100 ml of distilled water and was shakenvigorously to liberate the spores. The media was filtered through a milkfilter to separate hyphae from spores. The filtrate was centrifuged toseparate the spores from the liquid media. Repetitive suspension of theprecipitate with distillate water and recentrifugation proceeded toremove any traces of nutrient media (carbon source) from the spores. Thecollected spores were diluted by 1 ml of the salt liquid media. Sporeswere collected for each test organism. Equal volumes of the resultedspore suspension were mixed and about 1 ml of the fungus spores mixturewere prepared to be used for inoculation. The bacteria were directlyinoculated. Reserve stock of each microorganism was stored in therefrigerator. It should be renewed at least every four weeks andpreferably in between 10 and 20 days.

The developed mixed culture of microorganisms of the present inventioncan degrade numerous polyesters, including the thermoplastic main chainaliphatic polyester of the present invention, the thermoset heterochainpolyester of the present invention, polylactic acid (PLA),polycaprolactone (PCL), and polyglycolic acid (PGA), into biomass andrespiratory gases. The biodegradation of the thermoplastic main chainaliphatic polyester and the thermoset heterochain polyester can proceedto completion.

The structure of these polymers is the essential element of theirbiodegradability, and the percentage of hydrolyzable ester bonds on thepolymer affects biodegradation. The extent to which the thermosetheterochain polyester of the present invention is degradable relates tothe extent of crosslinking in the polyester.

The biodegradable polyesters of the present invention, the thermoplasticmain chain aliphatic polyester and the thermoset heterochain polyester,decompose into non-toxic compounds. The microorganisms can grow andutilize these polyesters as carbon sources for nutrition.

The following is an example of a method for degrading the polymers ofthe present invention using the mixed culture of microorganisms of thepresent invention. This example is not meant in any way to limit thescope of this invention.

EXAMPLE 13 A Method for Degrading Polymers Using a Mixed Culture ofMicroorganisms

In order to test the polymer toward microbial degradation, a fermenterfrom the submerged culture system was designed with suitable growthconditions. The degradation was estimated and modeled by quantifying theevolved CO₂ and the growing biomass. In this method, the test fungiconsisted of a mixture of Rhizopus chinesis, Rhizopus delemar,Penecillium pinophilum, Aspergillus niger, and Pseudomonas aeruginosa.

The carbon content of the polymers was transferred into respiratorygases and biomass growth. The weight loss in the polymer sample ismeasured with time. Respiratory activity of the degrading microorganismswas followed by analyzing the inlet and outlet gases coming to and fromthe cultivator flask. The inlet air stream was free of CO₂, for both thethermoplastic main chain aliphatic polyester and the thermosetheterochained polyester samples.

The thermoset heterochain aromatic polyester of the present inventionresisted biological degradation when contacted by the mixed culture ofmicroorganisms discussed above. It further resisted degradation whenPseudomonas cruciviae, Pseudomonas putida, and Corynebacteriumrestricta, all three of which are capable of metabolizing aromaticstructures, were added to the mixed culture. The thermoset heterochainaromatic polyester is a hard and brittle material due to the stiffnessassociated with the aromatic structure of the main chain. It appearedthat the chemical structure of the thermoset heterochain aromaticpolyester does not allow fungus adhesion followed by extracellularenzyme secretion, both of which are necessary for microbial degradation.

The polymers of the present invention may be used in consumer andindustrial products for making an assortment of plastics includingpackaging and packing materials. They provide advantages overconventional materials because of their degradability. The polymers ofthe present invention also have characteristics that make them suitablefor use as agricultural mulches and agricultural chemicals. Because boththe thermoplastic main chain and thermoset heterochain polyesters arecompletely degradable into respiratory gases and biomass without havinga toxic effect on the microbes in the mixed culture of microorganismsused to degrade them, it is reasonable to assume that the same resultscan be shown with other biological cells. Thus, the polymers of thepresent invention are ideally suited for use in biomedical applicationssuch as drug-delivery systems and surgical implants, because thesepolyesters decompose into non-toxic compounds. If used as adrug-delivery matrix, these polymers can be loaded with pharmaceuticalcompounds and inserted into a patient whereupon the polymer wouldharmlessly degrade and release the drug in a sustained and controlledmanner for a long period of time. These thermoplastic main chainaliphatic polyesters disclosed herein are similar in application topolylactic acid, a biodegradable polymer which is currently incommercial use as a time-release drug delivery matrix. The thermosetheterochain and thermoset heterochain aromatic polyesters of the presentinvention are similar in application to crosslinked polycaprolactone,another biodegradable polymer, which has shown potential for use insimilar biomedical applications.

From the foregoing, it will be seen that this invention is one welladapted to attain all the ends and objects hereinabove set forthtogether with other advantages that are obvious and inherent to thestructure. It will be understood that certain features andsubcombinations are of utility and may be employed without reference toother features and subcombinations. This is contemplated by and iswithin the scope of the claims. Since many possible embodiments may bemade of the invention without departing from the scope thereof, it is tobe understood that all matter herein set forth is to be interpreted asillustrative and not in a limiting sense.

What is claimed is:
 1. A process for degrading polyesters, comprising: treating a polyester formed from amine-protected glutamic acid with a biological catalyst so as to hydrolyze said polyester into monomers.
 2. A process as in claim 1, wherein said biological catalyst is lipase.
 3. A process for degrading polymers, comprising: treating polymers with a mixed culture of microorganisms comprised of the mixture of Rhizopus chinesis, Rhizopus delemar, Penecillium pinophilum, Aspergillus niger, and Pseudomonas aeruginosa, wherein said polymers are degraded into respiratory gases and biomass.
 4. A process as in claim 3, wherein said polymers are selected from the group consisting of a thermoplastic main chain aliphatic polyester, a thermoset heterochain polyester, polylactic acid (PLA), polycaprolactone (PCL), and polyglycolic acid (PGA). 